CD8+ T cells are critical components of the anti-viral immune response to viruses. Naive CD8+ T cells differentiate into effectors cells only after recognition of peptide-MHC Class I complexes on the surface of antigen presenting cells (APC). The peptides recognized as part of the peptide-MHC complex can be generated from two spatially distinct sources. Directly presented peptides are generated from protein antigens expressed within a virus-infected pAPC. Alternatively, pAPC that are not infected can present peptides, a process termed cross-priming. Cross-priming requires the transfer of antigen derived from virus-infected cells to pAPC prior to presentation to naive CD8+ T cells. The extent to which direct-priming or cross-priming contribute to activation of naive CD8+ T cells in vivo is not known. The overall objective of this project is to delineate the mechanisms used to generate MHC Class I-peptide complexes during priming of naive CD8+ T cells in vivo. Our underlying hypothesis is that alteration in protein expression or localization can, and does, define the usage of the direct or cross-priming pathways in vivo. To examine this issue we will use recombinant viruses to express multiple protein antigens and will analyze antigen presentation to naive and effector CD8+ T cells both in vitro and in vivo. In Aim 1 we will explore the mechanisms responsible for a novel mode of immune evasion in which late vaccinia virus antigens are prevented from entering the cross presentation pathway. In Aim 2 we will examine the mechanisms responsible for prolonged MHC Class I-restricted antigen presentation following clearance of virus, a prerequisite for optimal entry of CD8+ T cells in the memory phase of the response. We will examine the contribution of the direct and cross presentation pathways to the persistence of antigen presentation, and examine how a short-lived population of antigen presenting cells can present antigen for extended periods of time. In Aim 3 we will use a unique system to examine the efficiency of direct presentation by different cell types, and by cell types at various stages of maturation. In addition, we will definitely outline the role of direct and cross presentation in initiation of CD8+ T cell responses. Delineation of the mechanisms governing the use of different antigen presentation pathways in vivo is essential to provide a basis for the rational design of vaccines and immunotherapeutic strategies aimed at induction of protective CD8+ T cells.